The present invention relates to novel cationic amphiphilic compounds that facilitate the intracellular delivery of biologically active (therapeutic) molecules. The present invention relates also to pharmaceutical compositions that comprise such cationic amphiphiles, and that are useful to deliver into the cells of patients therapeutically effective amounts of biologically active molecules. The novel cationic amphiphilic compounds of the invention are particularly useful in relation to gene therapy.
Effective therapeutic use of many types of biologically active molecules has not been achieved simply because methods are not available to cause delivery of therapeutically effective amounts of such substances into the particular cells of a patient for which treatment therewith would provide therapeutic benefit. Efficient delivery of therapeutically sufficient amounts of such molecules into cells has often proved difficult, if not impossible, since, for example, the cell membrane presents a selectively-permeable barrier. Additionally, even when biologically active molecules successfully enter targeted cells, they may be degraded directly in the cell cytoplasm or even transported to structures in the the cell, such as lysosomal compartments, specialized for degradative processes. Thus both the nature of substances that are allowed to enter cells, and the amounts thereof that ultimately arrive at targeted locations within cells, at which they can provide therapeutic benefit, are strictly limited.
Although such selectivity is generally necessary in order that proper cell function can be maintained, it comes with the disadvantage that many therapeutically valuable substances (or therapeutically effective amounts thereof) are excluded. Additionally, the complex structure, behavior, and environment presented by an intact tissue that is targeted for intracellular delivery of biologically active molecules often interfere substantially with such delivery, in comparison with the case presented by populations of cells cultured in vitro.
Examples of biologically active molecules for which effective targeting to a patients"" tissues is often not achieved: (1) numerous proteins induding immunoglobin proteins, (2) polynucleotides such as genomic DNA, cDNA, or mRNA (3) antisense polynucleotides; and (4) many low molecular weight compounds, whether synthetic or naturally occurring, such as the peptide hormones and antibiotics.
One of the fundamental challenges now facing medical practicioners is that although the defective genes that are associated with numerous inherited diseases (or that represent disease risk factors including for various cancers) have been isolated and characterized, methods to correct the disease states themselves by providing patients with normal copies of such genes (the technique of gene therapy) are substantially lacking. Accordingly, the development of improved methods of intracellular delivery therefor is of great medical importance.
Examples of diseases that it is hoped can be treated by gene therapy include inherited disorders such as cystic fibrosis, Gaucher""s disease, Fabry""s disease, and muscular dystrophy. Representative of acquired disorders that can be treated are: (1) for cancersxe2x80x94multiple myeloma, leukemias, melanomas, ovarian carcinoma and small cell lung cancer; (2) for cardiovascular conditionsxe2x80x94progressive heart failure, restenosis, and hemophilias; and (3) for neurological conditionsxe2x80x94traumatic brain injury.
Gene therapy requires successful transfection of target cells in a patient. Transfection may generally be defined as the process of introducing an expressible polynudleotide (for example a gene, a CDNA, or an mRNA patterned thereon) into a cell. Successful expression of the encoding polynucleotide leads to production in the cells of a normal protein and leads to correction of the disease state associated with the abnormal gene. Therapies based on providing such proteins directly to target cells (protein replacement therapy) are often ineffective for the reasons mentioned above.
Cystic fibrosis, a common lethal genetic disorder, is a particular example of a disease that is a target for gene therapy. The disease is caused by the presence of one or more mutations in the gene that encodes a protein known as cystic fibrosis transmembrane conductance regulator (xe2x80x9cCFTRxe2x80x9d), and which regulates the movement of ions (and therefore fluid) across the cell membrane of epithelial cells, induding lung epithelial cells. Abnormal ion transport in airway cells leads to abnormal mucous secretion, inflammation and infection, tissue damage, and eventually death.
It is widely hoped that gene therapy will provide a long lasting and predictable form of therapy for certain disease states, and it is likely the only form of therapy suitable for many inhereted diseases. There remains however a critical need to develop compounds that faciliate entry of functional genes into cells, and whose activity in this regard is sufficient to provide for in vivo delivery of genes or other such biologically active therapeutic molecules in concentrations thereof that are sufficient for intracellular therapeutic effect.
In as much as compounds designed to facilitate intracellular delivery of biologically active molecules must interact with both non-polar and polar environments (in or on, for example, the plasma membrane, tissue fluids, compartments within the cell, and the biologically active molecule itself), such compounds are designed typically to contain both polar and non-polar domains. Compounds having both such domains may be termed amphiphiles, and many lipids and synthetic lipids that have been disclosed for use in facilitating such intracellular delivery (whether for in vitro or in vivo application) meet this definition. One particularly important class of such amphiphiles is the cationic amphiphiles. In general, cationic amphiphiles have polar groups that are capable of being positively charged at or around physiological pH, and this property is understood in the art to be important in defining how the amphiphiles interact with the many types of biologically active (therapeutic) molecules including, for example, negatively charged polynucleotides such as DNA.
Examples of cationic amphiphilic compounds that have both polar and non-polar domains and that are stated to be useful in relation to intracellular delivery of biologically active molecules are found, for example, in the following references, which contain also useful discussion of (1) the properties of such compounds that are understood in the art as making them suitable for such applications, and (2) the nature of structures, as understood in the art, that are formed by complexing of such amphiphiles with therapeutic molecules intended for intracellular delivery.
(1) Felgner, et al., Proc. Natl. Acad. Sci. USA, 84,7413-7417 (1987) disclose use of positively-charged synthetic cationic lipids including N-[1(2,3-dioleyloxy)propyl]-N,N,N-trimetylammonium chloride (xe2x80x9cDOTMAxe2x80x9d), to form lipid/DNA complexes suitable for transfections. See also Felgner et al., The Journal of Biological Chemisty, 269(4), 2550-2561 (1994).
(2) Behr et al., Proc. Natl. Acad. Sci. USA, 86,6982-6986 (1989) disclose numerous amphiphiles including dioctadecylamidologlycylspermine (xe2x80x9cDOGSxe2x80x9d).
(3) U.S. Pat. No. 5,283,185 to Epand et al. describes additional classes and species of amphiphiles including 3xcex2 [N-(N1,N1-dimethylaminoethane)-carbamoyl] cholesterol, termed xe2x80x9cDC-cholxe2x80x9d.
(4) Additional compounds that facilitate transport of biologically active molecules into cells are disclosed in U.S. Pat. No. 5,264,618 to Felgner et al. See also Felgner et al., The Journal Of Biological Chemistry, 269(4), pp. 2550-2561 (1994) for disclosure therein of further compounds including xe2x80x9cDMRIExe2x80x9d 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide, which is discussed below.
(5) Reference to amphiphiles suitable for intracellular delivery of biologically active molecules is also found in U.S. Pat. No. 5,334,761 to Gebeyehu et al., and in Felgner et al., Metods(Methods in Enzymology), 5,67-75 (1993).
Although the compounds mentioned in the above-identified references have been demonstrated to facilitate (although in many such cases only in vitro) the entry of biologically active molecules into cells, it is believed that the uptake efficiencies provided thereby are insufficient to support numerous therapeutic applications, particulary gene therapy. Additionally, since the above-identified compounds are understood to have only modest activity, substantial quantities thereof must be used leading to concerns about the toxicity of such compounds or of the metabolites thereof. Accordingly there is a need to develop a xe2x80x9csecond generationxe2x80x9d of cationic amphiphiles whose activity is so sufficient that successful therapies can be achieved therewith.
This invention provides for cationic amphiphiles that are particularly effective to facilitate transport of biologically active molecules into cells; The cationic amphiphiles of the invention are divided into four (4) groups, although it will be seen that there are certain structural and functional features that many of the amphiphiles share.
Accordingly, there are provided cationic amphiphiles of Group I (see FIG. 1, panels A, B, and C) capable of facilitating transport of biologically active molecules into cells, said amphiphiles having the structure (I), 
wherein:
Z is a steroid;
X is a carbon atom or a nitrogen atom;
Y is a short linking group, or Y is absent;
R3 is H, or a saturated or unsaturated aliphatic group;
R1 is xe2x80x94NHxe2x80x94, an alkylamine, or a polyalkylamine;
R4 is H, or a saturated or unsaturated aliphatic group;
R2 is xe2x80x94NHxe2x80x94, an alkylamine, or a polyalkylamine;
and wherein R1 is the same or is different from R2, except that both R1 and R2 cannot be xe2x80x94NHxe2x80x94.
In one preferred embodiment, the steroid component xe2x80x9cZxe2x80x9d is selected from the group consisting of 3-sterols, wherein said sterol molecule is linked by the 3-O-group thereof, or by Nxe2x80x94 in replacement thereof, to Y (or directly to X, if Y is absent). According to this aspect of the invention, particularly effective amphiphiles include, for example, spermidine cholesterol carbamate (N4-spermidine cholesteryl carbamate, amphiphile No. 53), and spermine cholesterol carbamate (N4-spermine cholesteryl carbamate, amphiphile No. 67), and amphiphiles patterned thereon.
In a further preferred embodiment, the steroid group is linked to Y (or directly to X, if Y is absent) from ring position 17 of the steroid nucleus (see FIGS. 1 and 22), or from the arm that normally extends from position 17 in many steroids (see the structure of cholesterol in FIG. 1), or from any shortened form of said arm.
In other preferred embodiments, within linking group Y are contained no more than about thee or four atoms that themselves form a bridge of covalent bonds between X and Z. In a specific preferred embodiment of the invention, Y is a linking group wherein no more than one atom of said group forms a bond with both X and Z, or Y is absent.
Representative amphiphiles provided according to Group I include: 
Additionally there are provided cationic amphiphiles of Group II (see FIG. 5) capable of facilitating transport of biologically active molecules into cells said amphiphiles having the structure (II), 
wherein:
Z is a steroid;
X is a carbon atom or a nitrogen atom;
Y is a linking group or Y is absent,
R3 is an amino acid, a derivatized amino acid, H or alkyl;
R1 is xe2x80x94NHxe2x80x94, an alkylamine, or a polyalkylamine;
R4 is an amino acid, a derivatized amino acid, H or alkyl;
R2 is xe2x80x94NHxe2x80x94, an alkylamine, or a polyalkylamine;
and wherein R1 is the same or is different from R2, except that both R1 and R2 cannot be xe2x80x94NHxe2x80x94.
Representative amphiphiles provided according to Group II include: 
Additionally there are provided cationic amphiphiles of Group III (see FIG. 6) capable of facilitating transport of biologically active molecules into cells said amphiphiles having the structure (III), 
wherein:
Z is an alkylamine or a dialkylamine, linked by the N-atom thereof, to Y (or directly to X, if Y is absent), wherein if Z is a dialkylamine, the alkyl groups thereof can be the same or different;
X is a carbon atom or a nitrogen atom;
Y is a short linking group, or Y is absent;
R3 is H, or a saturated or unsaturated aliphatic group;
R1 is xe2x80x94NHxe2x80x94, an alkylamine, or a polyalkylamine;
R4 is H, or a saturated or unsaturated aliphatic group;
R2 is xe2x80x94NHxe2x80x94, alkylamine, or a polyalkylamine;
and wherein R1 is the same or is different from R2, except that both R1 and R2 cannot be xe2x80x94NHxe2x80x94.
With respect to amphiphiles provided according to Structure (III), it is again preferred that within holding group Y there are contained no more than about three or four atoms that themselves form a bridge of covalent bonds between X and Z. In a specific preferred embodiment of the invention, Y is a linking group, such as  greater than C=O, wherein no more than one atom of said group forms a bond with both X and Z, or Y is absent.
Representative amphiphiles provided according to Group III include:. 
Additionally there are provided cationic amphiphiles of Group IV (see FIG. 7) capable of facilitating transport of biologically active molecules into cells said amphiphiles having the structure (IV), 
wherein:
A and B are independently O, N or S;
R5 and R6 are independently alkyl or acyl groups and may be saturated or contain sites of unsaturation;
C is selected from the group consisting of xe2x80x94CH2xe2x80x94,  greater than Cxe2x95x90O, and  greater than Cxe2x95x90S;
E is a carbon atom or a nitrogen atom;
D is a linking group such as xe2x80x94NH(Cxe2x95x90O)xe2x80x94 or xe2x80x94O(Cxe2x95x90O)xe2x80x94, or D is absent;
R3 is H, or a saturated or unsaturated aliphatic group;
R1 is xe2x80x94NHxe2x80x94, an alkylamine, or a polyalkylamine;
R4 is H, or a saturated or unsaturated aliphatic group;
R2 is xe2x80x94NHxe2x80x94, an alkylamine, or a polyalkylamine;
and wherein R1 is the same or is different from R2, except that both R1 and R2 cannot be xe2x80x94NHxe2x80x94.
Representative amphiphiles of Group IV include: 
The invention provides also for pharmaceutical compositions that comprise one or more cationic amphiphiles, and one or more biologically active molecules, wherein said compositions facilitate intracellular delivery in the tissues of patients of therapeutically effective amounts of the biologically active molecules. The pharmaceutical compositions of the invention may be formulated to contain one or more additional physiologically acceptable substances that stabilize the compositions for storage and/or contribute to the successful intracellular delivery of the biologically active molecules.
In a further aspect, the invention provides a method for facilitating the transfer of biologically active molecules into cells comprising the steps of preparing a dispersion of a cationic amphiphile of the invention; contacting said dispersion with a biologically active molecule to form a complex between said amphiphile and said molecule, and contacting cells with said complex thereby facilitating transfer of said biologically-active molecule into the cells.
For pharmaceutical use, the cationic amphiphile(s) of the invention may be formulated with one or more additional cationic amphiphiles induding those known in the art, or with neutral co-lipids such as dioleoylphosphatidylethanolamine, (xe2x80x9cDOPExe2x80x9d), to facilitate delivery to cells of the biologically active molecules. Additionally, compositions that comprise one or more cationic amphiphiles of the invention can be used to introduce biologically active molecules into plant cells, such as plant cells in tissue culture.
Additionally, the present application provides for novel plasmids suitable for complexing with the amphiphiles of the invention in order to treat patients by gene therapy, so that a high level of expression of the appropriate therapeutic transgene can be achieved. Representative examples thereof include the plasmid pCMVHI and pCFI. pCF1 plasmid contains the enhancer/promoter region from the immediate early gene of cytomegalovirus. The plamid also contains a hybrid intron located between the promoter and the transgene cDNA. The polyadenylation signal of the bovine growth hormone gene was selected for placement downstream from the transgene. These and other features contribute substantially to the improved transgene expression possible with this plasmid.
Further enhancements in plasmid performance are made possible by the provision of replicating episomal plasmids. Additional therapeutic enhancements are made possible by providing plasmids in which expression of the therapeutic transgene is placed under the control of a transcriptional promoter that is sensitive to the concentration of inflammation-related substances in the target tissue. Such plasmids are of particular use for the treatment of clinical cases in which inflammation is a major complication.
In a still further embodiment of the invention, particular organs or tissues may be targeted for gene therapy, by intravenous administration of amphiphile/transgene complexes, by adjusting the ratio of amphiphile to DNA in such complexes, and by adjusting the apparent charge or zeta potential thereof.
Further additional and representative aspects of the invention are described according to the Detailed Description of the Invention which follows directly.